Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum
Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have na...
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| creator | Serino-Silva, Caroline Morais-Zani, Karen Hikari Toyama, Marcos Toyama, Daniela de Oliveira Gaeta, Henrique Hessel Rodrigues, Caroline Fabri Bittencourt Aguiar, Wéslei da Silva Tashima, Alexandre Keiji Grego, Kathleen Fernandes Tanaka-Azevedo, Anita Mitico |
| description | Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation. |
| doi_str_mv | 10.1371/journal.pone.0193105 |
| format | Article |
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This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0193105</identifier><identifier>PMID: 29505564</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amino acids ; Biology and Life Sciences ; Bothrops ; Bothrops jararaca ; Circular dichroism ; Crotalus durissus terrificus ; Crotoxin ; Dichroism ; Edema ; Enzymatic activity ; Fatty acids ; Gel electrophoresis ; High performance liquid chromatography ; Inhibitors ; Liquid chromatography ; Mass spectrometry ; Mass spectroscopy ; Medicine and Health Sciences ; Membrane proteins ; Myonecrosis ; Neutralization ; Phospholipase ; Phospholipase A2 ; Phospholipids ; Plasma ; Proteins ; Purity ; Research and Analysis Methods ; Scientific imaging ; Snakes ; Sodium lauryl sulfate ; Spectroscopy ; Tropical diseases ; Venom ; Venom toxins ; Viperidae</subject><ispartof>PloS one, 2018-03, Vol.13 (3), p.e0193105-e0193105</ispartof><rights>2018 Serino-Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.</description><subject>Amino acids</subject><subject>Biology and Life Sciences</subject><subject>Bothrops</subject><subject>Bothrops jararaca</subject><subject>Circular dichroism</subject><subject>Crotalus durissus terrificus</subject><subject>Crotoxin</subject><subject>Dichroism</subject><subject>Edema</subject><subject>Enzymatic activity</subject><subject>Fatty acids</subject><subject>Gel electrophoresis</subject><subject>High performance liquid chromatography</subject><subject>Inhibitors</subject><subject>Liquid chromatography</subject><subject>Mass spectrometry</subject><subject>Mass spectroscopy</subject><subject>Medicine and Health Sciences</subject><subject>Membrane proteins</subject><subject>Myonecrosis</subject><subject>Neutralization</subject><subject>Phospholipase</subject><subject>Phospholipase A2</subject><subject>Phospholipids</subject><subject>Plasma</subject><subject>Proteins</subject><subject>Purity</subject><subject>Research and Analysis Methods</subject><subject>Scientific imaging</subject><subject>Snakes</subject><subject>Sodium lauryl sulfate</subject><subject>Spectroscopy</subject><subject>Tropical diseases</subject><subject>Venom</subject><subject>Venom 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and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum</title><author>Serino-Silva, Caroline ; Morais-Zani, Karen ; Hikari Toyama, Marcos ; Toyama, Daniela de Oliveira ; Gaeta, Henrique Hessel ; Rodrigues, Caroline Fabri Bittencourt ; Aguiar, Wéslei da Silva ; Tashima, Alexandre Keiji ; Grego, Kathleen Fernandes ; Tanaka-Azevedo, Anita Mitico</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c526t-95efe90483be9acaf1c1af9e06f7d0b878bfc1e8177147a2c54d97e947a9b2153</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Amino acids</topic><topic>Biology and Life Sciences</topic><topic>Bothrops</topic><topic>Bothrops jararaca</topic><topic>Circular dichroism</topic><topic>Crotalus durissus terrificus</topic><topic>Crotoxin</topic><topic>Dichroism</topic><topic>Edema</topic><topic>Enzymatic activity</topic><topic>Fatty acids</topic><topic>Gel electrophoresis</topic><topic>High performance liquid chromatography</topic><topic>Inhibitors</topic><topic>Liquid chromatography</topic><topic>Mass spectrometry</topic><topic>Mass spectroscopy</topic><topic>Medicine and Health Sciences</topic><topic>Membrane proteins</topic><topic>Myonecrosis</topic><topic>Neutralization</topic><topic>Phospholipase</topic><topic>Phospholipase A2</topic><topic>Phospholipids</topic><topic>Plasma</topic><topic>Proteins</topic><topic>Purity</topic><topic>Research and Analysis Methods</topic><topic>Scientific imaging</topic><topic>Snakes</topic><topic>Sodium lauryl sulfate</topic><topic>Spectroscopy</topic><topic>Tropical diseases</topic><topic>Venom</topic><topic>Venom toxins</topic><topic>Viperidae</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Serino-Silva, Caroline</creatorcontrib><creatorcontrib>Morais-Zani, Karen</creatorcontrib><creatorcontrib>Hikari Toyama, 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Karen</au><au>Hikari Toyama, Marcos</au><au>Toyama, Daniela de Oliveira</au><au>Gaeta, Henrique Hessel</au><au>Rodrigues, Caroline Fabri Bittencourt</au><au>Aguiar, Wéslei da Silva</au><au>Tashima, Alexandre Keiji</au><au>Grego, Kathleen Fernandes</au><au>Tanaka-Azevedo, Anita Mitico</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2018-03-05</date><risdate>2018</risdate><volume>13</volume><issue>3</issue><spage>e0193105</spage><epage>e0193105</epage><pages>e0193105-e0193105</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>29505564</pmid><doi>10.1371/journal.pone.0193105</doi><orcidid>https://orcid.org/0000-0001-9363-3752</orcidid><oa>free_for_read</oa></addata></record> |
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| issn | 1932-6203 1932-6203 |
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| source | DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Amino acids Biology and Life Sciences Bothrops Bothrops jararaca Circular dichroism Crotalus durissus terrificus Crotoxin Dichroism Edema Enzymatic activity Fatty acids Gel electrophoresis High performance liquid chromatography Inhibitors Liquid chromatography Mass spectrometry Mass spectroscopy Medicine and Health Sciences Membrane proteins Myonecrosis Neutralization Phospholipase Phospholipase A2 Phospholipids Plasma Proteins Purity Research and Analysis Methods Scientific imaging Snakes Sodium lauryl sulfate Spectroscopy Tropical diseases Venom Venom toxins Viperidae |
| title | Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-10T00%3A09%3A34IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Purification%20and%20characterization%20of%20the%20first%20%CE%B3-phospholipase%20inhibitor%20(%CE%B3PLI)%20from%20Bothrops%20jararaca%20snake%20serum&rft.jtitle=PloS%20one&rft.au=Serino-Silva,%20Caroline&rft.date=2018-03-05&rft.volume=13&rft.issue=3&rft.spage=e0193105&rft.epage=e0193105&rft.pages=e0193105-e0193105&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0193105&rft_dat=%3Cproquest_plos_%3E2011274150%3C/proquest_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2010822263&rft_id=info:pmid/29505564&rft_doaj_id=oai_doaj_org_article_29c81f3775464ed398fc2ca96bf2b1e5&rfr_iscdi=true |